The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.